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Abstract:

A microfluidic system comprising a microchannel, a carrier fluid
comprising a fluorinated oil in the microchannel, at least one plug
comprising an aqueous plug-fluid in the microchannel and substantially
surrounded on all sides by the carrier-fluid, and a fluorinated
surfactant comprising a functional group capable of selectively binding a
target molecule is disclosed. A compound for use therewith and a method
of synthesizing a fluorinated surfactant are also provided.

Claims:

1. A microfluidic system comprising: a microchannel; a carrier fluid
comprising a fluorinated oil in the microchannel; at least one plug
comprising an aqueous plug-fluid in the microchannel and substantially
surrounded on all sides by the carrier-fluid; a fluorinated surfactant
comprising a functional group capable of selectively binding a target
molecule.

2. The microfluidic system of claim 1, further comprising a second
fluorinated surfactant.

3. The microfluidic system of claim 2, wherein the second fluorinated
surfactant comprises a functional group that does not significantly bind
to the target molecule.

4. The microfluidic system of claim 1, wherein the target molecule is a
biological molecule.

5. The microfluidic system of claim 1, wherein the target molecule is
water soluble.

6. The microfluidic system of claim 1, wherein the functional group is
selected from the group comprising a nitrilotriacetate, an iminediacetate
a triazacyclononane, a biotin derivative, a glutathione derivative, a
maltose derivative, an antibody, an aptamer, a thioredoxin tag, a FLAG
tag, a hemaglutinin tag, and an OmpA signal sequence tag.

7. A compound of the formula ##STR00003## and salts thereof.

8. A method of synthesizing a fluorinated surfactant of the formula
CF3(CF2)n(CH2)mO(CH2CH2O)pH;
wherein n is an integer from 1 to 20; m is an integer from 1 to 4; p is
an integer from 3 to 6; comprising coupling a compound of the formula
CF3(CF2)n(CH2)mOH with a compound of the formula
HO(CH2CH2O)pH.

Description:

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 61/335,570, incorporated by reference herein in its
entirety.

TECHNICAL FIELD

[0003] The present disclosure relates to microfluidic systems, and in
particular, to interfaces that eliminate non-specific adsorption and
introduce specific interactions.

BACKGROUND

[0004] Interfaces are critical to consider when using biological
solutions. Biofouling, nonspecific adsorption and denaturing of proteins,
peptides, cells and other biological species can interfere with "normal"
interactions. This can lead to observations, results, or conclusions that
are not relevant to in vivo situations where these interfaces are absent.
Developing a system that minimizes the types of interfaces exposed to the
system, and that is able to control the remaining interfaces is
desirable.

BRIEF SUMMARY

[0005] In one aspect, a microfluidic system includes a microchannel, a
carrier fluid comprising a fluorinated oil in the microchannel, at least
one plug comprising an aqueous plug-fluid in the microchannel and
substantially surrounded on all sides by the carrier-fluid, and a
fluorinated surfactant comprising a functional group capable of
selectively binding a target molecule.

[0006] In some embodiments, the microfluidic system includes a second
fluorinated surfactant. The second fluorinated surfactant may have a
functional group that does not significantly bind to the target molecule.

[0007] In some embodiments, the target molecule is a biological molecule.

[0008] In some embodiments, the target molecule is water soluble.

[0009] In some embodiments, the functional group of the fluorinated
surfactant is selected from a nitrilotriacetate, an iminediacetate a
triazacyclononane, a biotin derivative, a glutathione derivative, a
maltose derivative, a thioredoxin tag, a FLAG tag, a hemaglutinin tag,
and an OmpA signal sequence tag.

[0010] In another aspect, a compound of the formula

##STR00001##

and salts thereof are disclosed.

[0011] In another aspect, a method of synthesizing a fluorinated
surfactant of the formula
CF3(CF2)n(CH2)mO(CH2CH2O)pH,
where n is an integer from 1 to 20, m is an integer from 1 to 4, p is an
integer from 3 to 6 is disclosed. The fluorinated surfactant is prepared
by coupling a compound of the formula
CF3(CF2)n(CH2)mOH and a compound of the formula
HO(CH2CH2O)pH.

DETAILED DESCRIPTION

[0012] Microfluidic systems that minimize the types of interfaces exposed
in the system and which enable better control of those interfaces are
disclosed. By surrounding plugs in a microfluidic system with, for
example, fluorinated oil, all interfaces except the fluorous-aqueous
interface are removed. Fluorinated oils are preferred, as they have very
low solubility towards aqueous and organic components, leading to minimal
contamination and loss of material. In certain embodiments, coating the
fluorous-aqueous interface with specific surfactants provides control of
the surface chemistry and can reduce nonspecific adsorption and/or
introduce specific interface-target molecule interactions.

[0013] Nonspecific interactions can be reduced through the use of fluorous
surfactants, amphiphiles, or detergents, which may be referred to as
"Rf-inert", which can comprise a fluorinated tail, which may be referred
to as "Rf", and a head group that assembles to form an inert surface at
the fluorous-aqueous (F:A) interface that is resistant to adsorption of
biological species including, for example, proteins and cells. The tail,
Rf, can comprise, for example,
CF3(CF2)n(CH2)m--, where, for example, n is an
integer between 0 and 20 and m is an integer between 1 and 4, or
fluorinated ethers, such as Krytox fluorinated ethers, for example those
comprising 14 to 44 monomeric units, or Fomblin fluorinated ethers. Head
groups of Rf-inert can comprise, for example, a polyethylene glycol
group, an oligoethylene group, a zwitterion, such as
poly((3-(methacryloylamino)propyl)-dimethyl(3-sulfopropyl)ammonium
hydroxide (poly(MPDSAH)) (as described in Cho, W. K.; Kong, B. Y.; Choi,
I. S., Langmuir 2007, 23, 5678-5682), or materials, including sugars,
zwitterions and sorbitol derivatives, described in U.S. Pat. No.
7,494,714 and U.S. Pat. No. 7,276,286 and U.S. Pat. No. 6,972,196 U.S.
Ser. No. 11/124,022, incorporated by reference herein in their entirety,
permethylated D-Dulcitol-Derived or D-Mannitol-Derived Polymer (MPDME),
such as are described in Metzke, M.; Guan, Z., Biomacromolecules 2008, 9,
208-215, a mannitol group, a permethyl sorbitol group, tri(sarcosine) or
N-acetylpiperazine, as described in Ostuni, E.; Chapman, R. G.; Liang, M.
N.; Meluleni, G.; Pier, G.; Ingber, D. E.; Whitesides, G. M., Langmuir,
2001, 17, 6336-6343.

[0014] The present invention can be used with, for example, microfluidic
technology, including plug technology, such as is disclosed in U.S. Pat.
Nos. 7,129,091 and 7,655,470, and patent applications PCT/US08/71374,
PCT/US08/71370, PCT/US07/26028, PCT/US09/46255, U.S. Ser. No. 12/777,099,
U.S. 61/340,872, U.S. 61/335,570, U.S. 61/262,375, U.S. Ser. No.
12/670,739, U.S. Ser. No. 12/670,725, U.S. Ser. No. 12/520,027, U.S. Ser.
No. 12/162,763, U.S. Ser. No. 11/174,298, U.S. Ser. No. 10/765,718, and
U.S. Ser. No. 11/589,700 all incorporated by reference herein in their
entireties, or SlipChip technology, as disclosed in PCT/US2010/028316,
incorporated by reference herein in its entirety.

[0015] The present invention can be used in sensors to reduce or eliminate
nonspecific or background adsorption or interactions. Other sensor
technologies have used rinsing to eliminate or reduce background signal.
The ability to reduce or eliminate this undesired adsorption allows for
detection of weaker binding events and labile interactions that, for
example, processes involving washing or rinsing can miss. Exemplary
diagnostic systems to which the present invention can be applied are
described in U.S. patent application Ser. Nos. 12/678,014, 11/698,802 and
11/698,757, incorporated by reference herein in their entireties. Rinsing
can result in the loss of real interactions if those interactions are
weak or very labile. The background signal can be minimized by using an
inert interface, as is formed by the use of Rf-inert, and, optionally, a
specific interaction can be introduced that allows for selective
adsorption to the interface, increasing the analyte signal. Enhancement
of the analyte signal can be sufficient to remove the need for rinsing,
allowing the detection of important but weak or labile binding events
that rinsing or nonspecific adsorption can mask.

[0016] In certain embodiments of the invention, the high surface area to
volume ratio of plugs favorably enhances the distribution of molecules in
a plug to increase sensitivity. In certain preferred embodiments, the
solution is depleted of a target analyte and substantially all of the
target analyte is located at the plug surface, leading to a strong
signal. It will be apparent to one skilled in the art that the target
analyte can be bound to a fluorophore, for example a fluorophore
compatible with FRET, for detection. Other exemplary detection strategies
include having a fluorescently labeled capture agent complexed with a
quencher-labeled analyte, wherein when the analyte is present, quencher
is displaced and the label on the capture agent is detectable. In certain
embodiments, the long axis of a plug can be oriented perpendicular to the
detection source, increasing the effective path length for enhanced
detection. Manipulation of the plug geometry by manipulating the channel
geometry can enhance this effect. In certain embodiments, by altering
droplet geometry, for example, by sending a plug through a constriction,
an analyte-complexing surfactant may be selectively concentrated in
certain parts of the plug leading to concentration of the target molecule
and enhancement of the analyte signal. An analyte-complexing surfactant
can also be concentrated via plug flow. For example, during flow,
surfactant and thus the target analyte can be concentrated at the back of
the plug leading to enhanced signal.

[0017] Other applications include protein crystallization experiments in
which it is desirable to increase, control or reduce interface induced
nucleation. Other applications include: enzyme-based assays in which it
is desirable to prevent the denaturing of proteins; cell-based assays in
which exposure to a single biocompatible interface can improve cell
viability and eliminate or reduce unwanted interactions and side effects;
screening for interactions between amyloid proteins and potential drug
species, since, by avoiding aggregation induced by interfacial adsorption
one can scan systematically for molecules which alter and preferably
inhibit the intrinsic self-aggregation process of amyloid peptides and
proteins; studying interactions between amyloid proteins and other
biological species; studying interactions between Aβ protein and
other species by eliminating or reducing unnatural aggregation due to air
water interfaces or other artificial interfaces. Another application is
studying the interaction of Aβ with ganglioside clusters by
generating precise clusters of gangliosides within droplets, as
gangliosides have been shown to interact with Aβ, and clustering of
gangliosides in cholesterol rich lipid rafts is thought to be
biologically relevant. Identifying a critical cluster size can lead to
insights in the disease progression. Such experiments can also generate
specific Aβ species for, for example, studying their toxicity.
Potential drugs for the inhibition of the interaction between Aβ and
ganglioside clusters can also be screened using certain embodiments of
the present invention. Other interactions between amyloid proteins can
also be studied, for example potential interactions between Tau and
Aβ.

[0018] Certain embodiments of the present invention can be used to detect
signs of Alzheimer's disease in brain fluids. Proteomic and metabolic
changes in the brain due to amyloid diseases can be monitored by certain
embodiments of the present invention. For example, cerebrospinal fluid
(CSF) or interstitial fluid (ISF) can be sampled, and multiple analytical
assays, for example, ELISA assays, can be performed in parallel.

[0019] The present invention can be used in combination with a
chemistrode, which is disclosed in U.S. Ser. No. 12/737,058, incorporated
herein by reference in its entirety.

[0020] It will be apparent to one skilled in the art that any functional
groups that are able to bind and/or capture a specific species in
solution can be linked to a fluorinated tail such as Rf to generate
molecules which may be termed "Rf-capture". Such molecules can be used to
concentrate and orient such species at the fluorinated phase/aqueous
phase interface. Such fluorinated capturing components can be used in the
absence or presence of inert fluorinated surfactants.

[0021] Certain embodiments of the present invention can be used for PCR,
for example by using Rf-inert to reduce or eliminate deactivation of
enzymes or adsorption of DNA or primers.

[0022] It will be apparent to one skilled in the art that a number of
species can be used to link a capture moiety, such as a chelating group
capable of binding to a His tag, to a fluorinated tail. Preferably, the
capture species is kept sufficiently far from the fluorinated
phase/aqueous phase interface that the interface does not interfere with
binding (as might be the case with large proteins) and the binding
species still maintains some degree of freedom. Possible linking groups
for maintaining sufficient spacing include, but are not limited to, the
following, alone or in combination: oligoethylene glycol groups, for
example, oligoethylene glycol groups containing two to six glycol units;
a carbamate group, for example one derived from carbonyl diimidazole; an
acetate group, for example one derived from an α-bromoester; a
succinate group, such as one derived from succinic anhydride; and other
simple linkers such as an ether, an amine, an ester, an amide, an
α-hydroxy amide, an ω-carboxyamide or an aromatic group.

[0023] It will be apparent to one skilled in the art that "capture" groups
that can be linked to fluorinated tails can comprise, for example:
divalent cation binding groups, such as nitrilotriacetate (NTA, for
binding to His tags), iminediacetate (IDA), or triazacyclononane (TACN);
specific peptide or protein binding tags, such as biotin (for binding to
streptavidin or avidin derivatives), glutathione (for binding to proteins
or other substances linked to glutathione-S-transferase), maltose (for
binding to proteins or other substances linked to maltose binding
protein), thioredoxin tag, FLAG tag, hemaglutinin tag, OmpA signal
sequence tag and the like. Capture groups can comprise, for example,
protein or nucleic acid species, including antibodies, antigens,
aptamers, DNA, RNA, or other oligonucleotides or peptides. Capture groups
can also comprise sugars, including, for example gangliosides (for
example, GM1), maltose, glucose, sialic acid or galactose. It will be
apparent to one skilled in the art that each such capture group will be
chosen to capture a substance with an affinity for the chosen capture
group. Other capture agents (tags) and their uses that can be used as
part of the present invention are disclosed in U.S. application Ser. No.
12/415,988, incorporated by reference herein in its entirety. Capture
agents can also be peptides, proteins, antibodies or aptamers. Conjugates
of fluorinated groups and proteins are disclosed in U.S. patent
application Ser. Nos. 12/201,894, 11/520,182, and U.S. Pat. No.
5,055,562, herein incorporated by reference in their entirety. Aptamers
are disclosed in U.S. Pat. Nos. 7,700,759, 5,773,598, 5,496,938,
5,580,737, 5,654,151, 5,726,017, 5,786,462, 5,503,978, 6,028,186,
6,110,900, 6,124,449, 6,127,119, 6,140,490, 6,147,204, 6,168,778, and
6,171,795, 5,472,841, 5,567,588, 5,582,981, 5,637,459, 5,683,867,
5,705,337, 5,712,375, and 6,083,696 herein incorporated by reference in
their entirety.

[0024] Certain embodiments of the present invention can be used as
sensors. Selectively concentrating a substance at an interface with an
appropriate capture element can increase a detectable signal. For
example, a light-adsorbing interface can be aligned perpendicular to a
photo detector (e.g., a detector comprising microscope optics) providing
improved detection. The channel geometry that a plug passes through at
the point of detection can be altered to increase the detectability of a
signal. In certain embodiments, the mobility of the surfactants may allow
for selective concentration of Rf-capture when plug geometry is altered,
thus further increasing the detectability of a target molecule. For
example, by flowing plugs, fluorinated surfactants comprising capture
groups together with the bound target analyte can be concentrated at the
back of the plug, allowing for an increase in the detectability of a
signal. Such techniques may also use inert fluorinated surfactants to
minimize the background noise, for example by reducing denaturation, or
concentration of non-target species at the fluorinated phase/aqueous
phase interface.

[0025] Certain embodiments of the invention can be used for protein
crystallization. As described herein, surfactants comprising a capture
agent can be used to selectively and specifically concentrate and orient
a target analyte at the fluorinated phase/aqueous phase interface,
leading to an increased rate or chance of nucleation and improved crystal
quality.

[0026] Certain embodiments of the invention can be used to manipulate
protein aggregation. For example, this technique can be used to explore
interactions with specific surface chemistries. Gangliosides, for
example, have been shown to interact with Aβ. Functionalizing the
fluorinated phase/aqueous phase interface with gangliosides or other
species can provide information on how they affect Aβ aggregation.
In addition, certain embodiments of the invention can be used to
investigate whether conditions such as dialysis related amyloidosis (DRA)
are due to protein exposure to an unnatural surface, or if some critical
component is getting concentrated or depleted by the procedure. The
present invention provides the ability to cleanly control interfacial
interactions to study such systems.

[0027] Certain embodiments of the invention can be used for screening for
drug-peptide interactions. Drugs that inhibit protein-protein
interactions can be screened. One can use such embodiments to screen for
drugs that interfere with biologically relevant interactions that
facilitate, for example, the aggregation of Aβ or other amyloid
proteins.

[0028] Certain embodiments of the invention can be used for enzyme assays.
Some enzymes and proteins do not always perform their function in
solution and often are located or interact with other species at
membranes or other biological interfaces. The localization, concentration
and orientation that can result from this, as well as potential
allosteric interactions, can greatly alter the activity of an enzyme.
Thus studies that explore and account for these types of interactions are
more relevant to actual events in vivo than experiments done in bulk
solution (See Gureasko, J.; Galush, W. J.; Boykevisch, S.; Sondermann,
H.; Bar-Sagi, D.; Groves, J. T.; Kuriyan, J. Nat. Struct. Mol. Biol.
2008, 15, 452-461). The present invention can be used to precisely
control interactions at interfaces for this type of study.

[0029] Certain embodiments of the invention can be used for
multi-component structure formation. The assembly of multicomponent
structures at or in interfaces is critical for many systems including the
immune response and cellular signaling. Formation of the immunological
synapse is one example.

[0030] Certain embodiments of the invention can be used for cell-based or
bacterial assays, including selective activation of cells that have
certain binding affinities. All cells have functionalities that are
important for recognition and cellular function. Some cells need to
adhere to a surface to properly function. The adhesion in biological
systems is not always on a flat or immobile surface as is often the case
with other model systems, so cellular interactions with curved and/or
mobile interfaces are of interest.

[0031] Certain embodiments of the invention can be used to extract or
capture cells (for example, pathogens, circulating tumor cells, or immune
cells) or other biological species (for example, proteins, toxins, or
inflammatory cytokines) from biological samples such as blood or CSF,
using capture agents such as antibodies, antigens, nanobodies, aptamers,
and the like that are anchored with a fluorinated tail.

[0032] Certain embodiments of the invention can be used for nanoparticle
or other material synthesis. Fluorinated surfactants comprising capture
groups can serve as an organizing scaffold, nucleating, and/or
stabilizing agent to control formation of specific species, polymorphs or
surfaces.

[0033] Certain embodiments of the invention can be used for multiphase
transport, reaction or separation. For example, surfactant
functionalities can dictate the reactivity of plugs. If different
surfactants are localized to different plugs, then multistep reactions
can occur as reagents and/or intermediates are transported and/or
transferred between plugs using previously described techniques.

[0034] Certain embodiments of the invention can be used to create a
barrier using surfactant functionalities that permit certain species to
freely transport between plugs while other species are trapped. Such
control of permeability offers a system that mimics some components of
cellular behavior.

[0035] In certain embodiments of the present invention, a microfluidic
method is used to control interfacial chemistry in nanoliter droplets to
enable miniaturized in vitro measurements of protein aggregation.
Measuring aggregation of proteins experimentally is applicable to
understanding the biophysics of, for example, amyloidosis, and for
developing methods for detection and treatment of, for example, amyloid
diseases. In typical in vitro aggregation experiments, adsorption of
amyloid peptides to the air/water and other solid/water interfaces
accelerates peptide aggregation by enhancing nucleation. Intrinsic
stochasticity of nucleation phenomena implies that obtaining
statistically significant data often requires multiple experiments
performed in parallel, making miniaturization of these experiments
attractive. The interfacial effects, however, become even more pronounced
upon miniaturization of aggregation experiments, as miniaturization leads
to an increase of the surface-to-volume ratio. Miniaturization of
aggregation experiments is especially desirable for samples available
only in small volumes, such as cerebrospinal fluid (CSF) from, for
example, mice. CSF is known to contain components that inhibit formation
of amyloid aggregates, and the balance of inhibitory and pro-aggregation
activities changes with aging and progression of disease. Analysis of CSF
is therefore useful for biomarker discovery and monitoring effects of
drugs in animal models of Alzheimer's diseases. For mouse experiments,
such panels of experiments are not easily done with standard
multiwell-plate in vitro assays that require tens of microliters of
sample, given that the volume of CSF obtainable from a single mouse is
only a few microliters.

[0036] In order to miniaturize aggregation experiments while controlling
the interfacial chemistry one can use a plug-based microfluidic approach
in, for example, poly(dimethylsiloxane) (PDMS) microfluidic devices
modified with Teflon tubing. Exemplary plugs can comprise nanoliter
fluorocarbon-surrounded aqueous droplets formed in the flow of immiscible
fluids inside microfluidic channels. Once an amyloid peptide, for
example, Aβ40, is encapsulated inside a plug, it is protected
from the surfaces of microchannels by a layer of fluorocarbon, and the
surface chemistry of the aqueous-fluorous interface, rather than the
aqueous-channel interface, becomes relevant to the peptide. It was
demonstrated that the adsorption of Aβ40 to the
aqueous-fluorous interface can be minimized by comparing the behavior of
Aβ40 labeled with HiLyte-488 at the N-terminus at two
liquid/liquid interfaces: (i) an aqueous peptide/fluorocarbon interface
without surfactant and (ii) an aqueous
peptide/n-C8F17CH2-OEG3-protected fluorocarbon
interface. n-C8F17CH2-OEG3 is an amphiphilic
fluorinated surfactant,
CF3(CF2)7CH2O(CH2CH2O)3H, that can be
added to the carrier fluid, and without wishing to be bound by theory, is
thought to assemble spontaneously at the aqueous-fluorous interface, and
present triethyleneglycol groups to the aqueous phase, thereby preventing
or reducing protein adsorption.

[0037] In plugs containing no n-C8F17CH2-OEG3, a
fluorescence signal of the labeled Aβ40 peptide increased at
the plug edges, indicating adsorption of the peptide at the interface 2
hours after encapsulation. By contrast, in plugs with
n-C8F17CH2-OEG3, the fluorescence signal of the
labeled peptide was evenly distributed, demonstrating that
n-C8F17CH2-OEG3 prevents Aβ40 adsorption to
the interface. The effectiveness of n-C8F17CH2-OEG3
was a function of its concentration in the carrier fluid. To decrease
exposure of Aβ40 to the channel walls prior to plug formation,
one can insert Teflon tubing into the peptide inlet. To confirm that
Aβ40 is not lost during the plug formation process, the
fluorescence intensity of 5-40 μM fluorescein-labeled Aβ40
was measured after encapsulation within the plugs containing
n-C8F17CH2-OEG3. A linear increase in intensity that
coincided with the increase of intensity of fluorescein (combined
R2=0.99), a compound that does not adsorb to surfaces in our
devices, was found.

[0038] Certain embodiments of the present invention can be used to monitor
aggregation kinetics. For example, the differences in the aggregation
kinetics of Aβ40 in plugs with and without
n-C8F17CH2-OEG3 were monitored using Thioflavin T
(ThT), a widely used dye that has been used in other systems to monitor
aggregation by observing the increase of ThT fluorescence upon binding to
amyloid aggregates. The aggregation kinetics of Aβ40 depend on
nucleation and are typically described by a sigmoidal curve. As a
control, 50 μM Aβ40 aggregation kinetics in 100 μL
volumes in a well plate were monitored. A lag period of ˜10 hours
was observed before the ThT fluorescence indicated the start of
Aβ40 aggregation. In plugs with a fluorocarbon/water interface,
the lag time of the aggregation reaction shortened to ˜2 hours,
correlating with the adsorption of the peptide to the fluorocarbon/water
interface and also with the increased surface-to-volume ratio in 10 nL
volumes. In contrast, the lag time for aggregation of Aβ40 in
plugs with n-C8F17CH2-OEG3 present increased to many
days. After 250 hours, only 3 plugs out of 15 showed evidence of
Aβ40 aggregation. Even after 2 months, only 8 plugs out of 15
showed evidence of Aβ40 aggregation. Thus, control of the
interfacial chemistry decreases the aggregation kinetics of
Aβ40 by at least an order of magnitude.

[0039] It has been shown that the system reliably handles aggregation
experiments with small volumes using CSF from a single mouse (5 μL).
The relative ability of CSF sampled from two mouse strains to inhibit
Aβ40 aggregation was also studied. In this comparison,
nontransgenic (wt) mice and ceAPPswePS1ΔE9/TTR-/- transgenic mice
that exhibit amyloid deposition throughout the cortex and hippocampus
were used. The ceAPPswePS1ΔE9/TTR-/- mice lack the gene encoding
transthyretin (TTR), a molecule that is enriched in CSF and known to
associate with Aβ40 in both in vitro and in vivo settings. The
inhibitory potency of CSF for each mouse was tested by generating plugs
containing buffer, ThT, Aβ40 and a given concentration of CSF.
Titrations were performed by changing the relative flow rates of all
inlet streams to generate plugs containing constant concentrations of
ions and buffer, ThT and Aβ40, but different concentrations of
CSF. For each of the 15 different concentrations of CSF, 50 plugs were
generated, giving 750 experiments for each sample of CSF. Aβ40
aggregation was then monitored by the increase of ThT fluorescence. In
contrast to the experiments with low ionic strength, the ionic strength
of the buffer solution in CSF experiments was adjusted to accelerate
aggregation and to resemble the ion composition of mouse CSF, which
includes a high concentration of Ca2+ and Mg2+ ions. In accord
with previous reports, the higher ionic strength led to a decrease in the
lag time of aggregation of Aβ. CSF from the wild type mouse was able
to inhibit Aβ40 aggregation when added at concentrations higher
than 1:25 dilution. On the other hand, CSF from the
ceAPPswePS1ΔE9/TTR-/- mice did not display significant inhibitory
effect on Aβ40 aggregation at concentrations as high as 1:5
dilutions, suggesting that TTR plays a role in inhibiting Aβ40
aggregation. These results are consistent with the difference in the
inhibitory potency of human CSF from patients with and without
Alzheimer's disease. Thus, the plug-based system has the capability to
analyze volume-limited samples from mice. Plug-based microfluidics and
control of surface chemistry can be used to miniaturize peptide
aggregation experiments to nanoliter volumes. As will be apparent to one
skilled in the art, plug-based microfluidics are compatible with other
analytical methods potentially applicable to analysis of aggregation,
including, but not limited to, mass spectrometry and fluorescence
correlation spectroscopy. Certain embodiments of the present invention
are useful for studying, for example, in vitro aggregation biophysics,
for example time-controlled aging and nucleation-growth experiments of
amyloid peptides. Certain embodiments of the present invention are useful
for diagnostics or for monitoring potential treatments, for example
through repeated analysis of CSF or brain interstitial fluid from the
same live animal (including humans), for example using a chemistrode.

[0040] It will be apparent to one skilled in the art that the present
invention can be used with any peptides or protein that are unstable in
structure, especially amphiphilic peptides or proteins that are
susceptible to adsorption to interfaces.

[0041] One embodiment of the present invention is a method of synthesizing
a fluorinated surfactant of the structure
CF3(CF2)n(CH2)mO(CH2CH2O)pH,
wherein n=1 to 20, m=1 to 4 and p=3 to 6, comprising coupling
CF3(CF2)n(CH2)mOH and
HO(CH2CH2O)pH catalyzed by a trialkyl phosphine and a
reagent of the form R(CO)NN(CO)R, for example where R is piperidine. It
will be apparent to one skilled in the art that other reagents, including
other reagents used to carry out Mitsunobo reactions, can be used for the
coupling.

[0042] Certain embodiments of the present invention can be used to
generate functionalizable, mobile interfaces in plug-based microfluidics.
Control of interfaces is advancing studies of biological interfaces,
heterogeneous reactions and nanotechnology. Self-assembled monolayers
(SAMs) have been useful for such studies, however SAMs are not laterally
mobile, and therefore less applicable to systems where motion along the
interface is important, such as in protein crystallization or when
multiple membrane-associated proteins must assemble to perform their
function. Lipid-based methods such as monolayers, vesicles, black lipid
membranes and supported lipid bilayers (SLBs) are typically mobile and
are widely used, but they are typically less robust and stable than SAMs
and increasing the throughput capacity of such experiments remains a work
in progress. Lipid-based methods effectively form 2-D crystals of
proteins, but successes with nucleation of 3-D crystals are rare,
presumably because salts, PEG, and detergents typically used in
crystallization experiments perturb lipid structures. Furthermore, using
these methods it is difficult to set up the hundreds of experiments
necessary for crystallization screening.

[0043] Plug-based microfluidic systems provide the ability to generate
thousands of unique reaction mixtures as droplets surrounded by
fluorocarbon, allowing for rapid and expansive exploration of chemical
space. In certain embodiments, His-tag binding chemistry can be
implemented in an RfNTA molecule, which causes specific adsorption of a
protein at an interface, offering interfacial functionality and mobility.
Certain embodiments of this invention can be used for the crystallization
of a His-tagged reaction center, performing multiple (for example, tens,
hundreds, thousands, tens of thousands, or hundreds of thousands)
crystallization trials, thereby increasing the range of crystal producing
conditions, the success rate at a given condition, the rate of
nucleation, and/or the quality of the crystal formed.

[0044] A His-tagged green fluorescent protein (hGFP) can be used to
demonstrate that RfNTA with Ni2+ (RfNTA:Ni) introduces specific
interactions with His-tagged proteins at the plug interface. Alone, hGFP
is uniformly distributed in the plug; with RfNTA:Ni, hGFP is concentrated
at the plug-carrier fluid interface. This specific interaction takes
place in the presence of other surfactants. Addition of
n-C8F17CH2-OEG3 does not inhibit hGFP interfacial
adsorption. Hydrocarbon detergents that solubilize membrane protein do
not compete with RfNTA for the aqueous-fluorous interface and do not
interfere with the surface specific interaction. All experiments
contained 0.05% w/v lauryldimethylamine N-oxide (LDAO).

[0045] Control experiments showed that interfacial adsorption depends on
the formation of an RfNTA:Ni:hGFP complex. When adding (a) only
Ni2+, (b) only RfNTA, or (c) a complex of Ni2+ and NTA, with no
fluorous tail, to hGFP, the fluorescence was uniform across the plug.
Addition of EDTA (12 mM) or imidazole (120 mM) disrupted interfacial
adsorption, consistent with EDTA binding tightly to Ni2+, preventing
formation of the RfNTA:Ni:hGFP complex and excess imidazole outcompeting
His-tagged proteins for interaction with RfNTA:Ni.

[0046] The functionalized interface remains mobile, as shown by
fluorescence recovery after photobleaching (FRAP) experiments. The rate
of recovery is consistent with a diffusion coefficient
D0=1.1-3.1×10-12 m2/sec, lower than for simple
diffusion (D0=9×10-11 m2/sec), but higher than for
diffusion in lipid monolayers or bilayers (D0=1.3×10-13
m2/sec), presumably because the less densely packed interface was
more fluid. Decreasing the packing density increased the rate of
recovery.

[0047] RfNTA:Ni can be used under harsh conditions such as those
encountered during protein crystallization because, like other
fluorinated molecules containing NTA, such as those disclosed in U.S.
Ser. No. 10/576,767, incorporated herein by reference in its entirety, it
resists interference by detergents. RfNTA:Ni can aid crystallization in
at least the following ways: (1) by increasing the range of conditions
for crystallization and the success rate at a given condition, for
example by lowering the nucleation barrier; (2) by increasing the rate of
protein crystallization, for example by forming nuclei more quickly; (3)
by improving crystal quality, for example by increasing order via the
orienting effect at the interface.

[0048] The His-tagged reaction center (hRC) from Rhodobacter sphaeroides
has been examined. Using previously known techniques, crystals
irreproducibly form over 5 days at a high precipitant concentration. With
only Ni2+ added, all nucleation is suppressed, indicating that
Ni2+ inhibits crystal formation. With only RfNTA added, the number
of plugs with crystals and the quality of crystals did not substantially
change, but crystals formed more rapidly, possibly due to a residual
amount of divalent cation present in the solution. When both RfNTA and
Ni2+ were added, the number of nucleation events increased
significantly and crystals formed within 1 day. In addition,
crystallization occurred at lower concentrations of precipitant, and the
quality of diffraction increased. For high concentrations of RfNTA:Ni at
the highest concentration of precipitant, formation of crystals actually
decreased. Without wishing to be bound by theory, this may be due to
either interference from nonspecific adsorption through surface
histidines or to nucleation occurring too rapidly to allow for ordered
growth. When 10 mM imidazole was added the rate of crystal formation, the
number of crystals, and the quality of crystals were improved.

[0049] The present invention can be used for the rapid generation of
mobile, functionalized interfaces for exploring large areas of chemical
space, both in solution and at the interface. Surface composition can be
varied by changing the tagging functionalities of surfactants at the
interface, or by varying the tagged molecules in solution. Such
variations allow application of this method to other processes, such as
nucleation of amyloid protein aggregates, multicomponent interfacial
assembly, and capture assays that rely on binding to the interface and do
not require washing steps to detect binding. Additional analytical
techniques compatible with plugs, such as in-situ x-ray diffraction,
mass-spectrometry and fluorescence correlation spectroscopy expand the
applicability of the method.

Methods

Fabrication of a Microfluidic Device for Aggregation Experiments.

[0050] Microchannels were fabricated as disclosed in U.S. Pat. No.
6,767,194, U.S. Pat. No. 7,323,143 and U.S. Pat. No. 6,645,432, herein
incorporated by reference in their entirety. Briefly, microchannels with
rectangular cross section were obtained by rapid prototyping in
poly(dimethylsiloxane) (PDMS). The PDMS/PDMS devices were sealed using a
Plasma Prep II plasma cleaner. After sealing, the microchannels were
rendered hydrophobic by baking the PDMS devices in a 120° C. oven
for over one hour.

ThT Aggregation Assay.

[0051] Aβ40
(NH2-DAEFR5HDSGY10EVHHQ15KLVFF20AEDVG25SNKG-
A30IIGLM35VGGVV40--COOH) was obtained from rPeptide. HiLyte
and fluorescein labeled Aβ40 were obtained from Anaspec. To
dissolve preformed peptide aggregates of Aβ40, the lyophilized
peptide was first dissolved in hexafluoroisopropanol (HFIP). The HFIP was
evaporated from the peptides samples and the samples were then dried
under high vacuum overnight. The concentration of the peptide solution
was determined by absorbance at 280 nm using an extinction coefficient of
ε=1360 M-1 cm-1 for Tyr. Aβ40 was dissolved
in water shortly before use. Aggregation of Aβ40 was detected
by Thioflavin T (ThT) fluorescence. Aggregation experiments in a well
plate were done on a Polarstar Omega (BMG Labtech). For this, 50 μM
Aβ40 and 50 μM ThT dissolved in 50 mM Tris (pH 7.3) at
37° C. was used.

Microfluidic Experiment.

[0052] Aqueous and fluorous solutions were filled into 10 μL gastight
syringes (Hamilton Company), which were then connected to the
microfluidic device by 30-gauge Teflon tubing (Weico-Wire & Cable). For
Aβ40 and CSF solutions, syringes were filled with water and
aspirated 2 μL of either solution into Teflon tubing, which were than
connected to a syringe and PDSM device. Syringes were driven by PHD 2000
Infusion pumps (Harvard Apparatus). Fluorinert FC-40 or FC-70 (3M),
purified by distillation before use, was used as a carrier fluid. The
fluoro-surfactant
CF3(CF2)7CH2O(CH2CH2O)3H
(n-C8F17CH2-OEG3) was synthesized as described
herein.

Determination of the Concentration of Aβ40 in Plugs.

[0053] Various concentrations of fluorescein-labeled Aβ40 were
encapsulated in plugs. Additionally, a standard curve with fluorescein
(dissolved in 0.05% NH4OH) encapsulated in plugs was recorded. A
linear behavior of the fluorescence intensity of Aβ40 in plugs
with increasing Aβ40 concentration was observed (R2 value
0.99). Furthermore, the fluorescence intensities of the peptide in the
plugs were in agreement with the standard curve, indicating that
Aβ40 was not lost during the encapsulation process. The
absolute fluorescence intensity was obtained using a Lecia DMI 6000B
epi-fluorescence microscope with a 10×0.4 objective coupled to a
Hamamatsu ORCA ERG 1394 CCD camera with an exposure time of 3 ms. This
procedure was used for both the fluorescein standard curve and the
labeled Aβ40.

In Vitro Aggregation Experiments in Plugs.

[0054] Experiments were performed with a four-channel inlet device.
Aqueous Aβ40, Tris buffer, and a solution of ThT were loaded
into syringes, and syringes were connected to the convening channels with
cross sectional diameters of 225×200 μm2 of a microfluidic
device. The final concentrations of Aβ40, ThT, and Tris (pH 7.3
at 37° C.) in plugs were always 50 μM, 50 μM and 50 mM,
respectively. A syringe containing water-immiscible fluorocarbon FC-70
without or with 0.75 mg/ml n-C8F17CH2-OEG3 was
connected to a perpendicular channel. The flow of the aqueous solutions
and fluorocarbon was established by driving syringes with syringe pumps.
Typical flow rates for the total aqueous and fluorocarbon phase were 0.8
and 1.2 μL/min respectively. Plugs were collected in Teflon tubing
with an inner diameter of 200 μm. After the last plug was formed, the
syringes were disconnected and the flow was stopped. To avoid evaporation
of water from the plugs during the experiments, the plugs within the
Teflon tubing were inserted into oil filled glass capillaries and the
capillaries were sealed with sealing wax. The capillaries were incubated
at constant temperature (37° C.) and the ThT fluorescence of the
plugs was measured periodically.

Cerebrospinal Fluid Experiments.

[0055] CSF was isolated from the cisterna magna compartment as previously
described in DeMattos, et al., J. Neurochem. 2002, 81, 229. In brief,
mice were anesthetized with a mixture of ketamine and xylene. An incision
was made from the top of the skull to the dorsal thorax, and the
musculature was removed from the base of the skull to the first vertebrae
to expose the meninges overlying the cisterna magna. The tissue above the
cisterna magna was excised. A microneedle was used to punctuate the
arachnoid membrane covering the cistern. The CSF, which is under positive
pressure as a result of blood pressure, respiration and positioning of
the animal, began to flow out the needle entry site once the needle was
removed, and was collected using a polypropylene narrow bore pipette as
it exited the compartment. Experiments with mouse CSF were done using a
five-channel inlet device, where (1) Aβ40, (2) buffer, (3) ThT
in a salt solution, (4) CSF, and (5) FC-40 with 1.25 mg/ml
n-C8F17CH2-OEG3, were each allocated to one channel.
In order to screen the inhibitory potency of CSF on Aβ40, three
experiments were performed for each mouse CSF sample with three different
pre-dilutions factors of CSF (1:10, 1:5 and 1:1). Dilutions of CSF were
done with a buffer containing 150 mM NaCl and 10 mM PO42- at pH
7.3 and 37° C. Further dilutions (1:20, 1:10, 1:5, 1:4, and 1:2.5)
of the pre-diluted samples were obtained by changing the flow rates of
the aqueous streams. The flow rate ratio of the total aqueous and FC-40
phase was kept constant (2 μL min-1/2 μL min-1). The flow
rates of the Aβ40 and ThT/salt solutions were also kept
constant. Only the buffer and CSF streams were changed to counteract each
other. The final concentration of both Aβ40 and ThT in the
plugs was 50 μM. The salt concentration added to the ThT solution was
adjusted to obtain a salt composition of 150 mM NaCl, 3 mM KCl, 0.8 mM
MgCl2, 1.4 mM CaCl2, and 10 mM
NaH2PO4/Na2HPO4 pH 7.3 at 37° C., in a plug.
Plugs were stored in Teflon tubing at 37° C. and fluorescence
images of the plugs were taken periodically. For each CSF concentration
series, 50 plugs were formed and of those 15 were monitored. The
fluorescence intensity of the 15 plugs were monitored. The aggregation
kinetics of Aβ40 in dilutions of the above described buffer
(1:1, 1:2, and 1:4) were monitored, showing that increasing the ionic
strength of the buffer is responsible for nucleating Aβ40 in
plugs without a lag time.

Data Evaluation.

[0056] The fluorescence intensity in plugs was measured by taking
fluorescence micrographs of single plugs using a Leica DMI 6000
microscope with a cooled CCD camera ORCA ERG 1394 (12-bit 1344×1024
resolution) (Hamamatsu Photonics). A MetaMorph Imaging System version
6.1r3 (Universal Imaging Corp) was used for imaging acquisition and
Matlab 7a (Mathworks) for image analysis. The fluorescence intensity was
extracted from the images by calculating the average pixel intensity of
the plugs at each time point. For comparison of the experiments in plugs
and well plate the ThT fluorescence intensity was normalized to the
maximal intensity at the end point of the measurement,
I-I0/Imax, where I, I0 and Imax are the fluorescence
intensity, fluorescence intensity at time point 0, and the maximal
fluorescence intensity at the end point of the measurement, respectively.
In CSF experiments the ThT fluorescence signal was normalized to the
starting value, I-I0, were I0 is the average fluorescence
signal determined from 15 plugs.

n-C8F17CH2-OEG3 Concentration Assay.

[0057] To avoid Aβ40 adsorption to the plug interface, it is
preferable to use the correct concentration of
n-C8F17CH2-OEG3 in the carrier fluid. It was found
that a concentration lower than 0.5 mg/ml in FC-70 is less preferred due
to Aβ40 adsorption to the plug interface. For the carrier fluid
FC-40 Aβ40 adsorption can be avoided by using concentrations of
n-C8F17CH2-OEG3 above 0.75 mg/ml. Therefore
saturation of the plug surface with surfactant molecules is dependent on
the carrier fluid. n-C8F17CH2-OEG3 concentrations
above 1 and 1.5 mg/ml, however, lead to aqueous micelle formation and
plug coalescence in FC-70 and FC-40, respectively. ThT fluorescence in
plugs formed in the stream of FC-70 with aggregated Aβ40 after
250 hours is distributed over the whole plug volume, whereas without
n-C8F17CH2-OEG3 or at low concentrations of
n-C8F17CH2-OEG3 the fluorescence signal is located at
the plug interface. Without wishing to be bound by theory, this finding
supports that aggregation of Aβ40 is caused by adsorption to
unfavorable interfaces.

[0059] Triethylene glycol (Acros, MW: 150.17) was dried over molecular
sieves and 1 equivalent was added to a dried round bottom flask with stir
bar. 1.5 equivalents of CF3(CF2)7CH2OH
(Sigma-Aldrich, MW: 450.1) and 1.3 equivalents of
1,1'-azodicarbonyldipiperidine (ADDP) (TCI, MW: 252.31) were also added
to the flask. These were dissolved in anhydrous benzene (Sigma-Aldrich),
so that concentrations are around 0.1 M. The reaction mixture was then
stirred and heated to about 60° C. Then 1.4 equivalents of
tributylphosphine (Sigma-Aldrich, MW: 202.32) were added to benzene
(about half the volume that was added to the round bottom flask) in an
addition funnel. This solution was then slowly added to the reaction
mixture over 1-2 hours. Once addition was complete the mixture was heated
for an additional 3 hours, or until color was lost. Upon cooling, a
significant amount of precipitant formed. The precipitant was filtered
off and solvent removed by rotary-evaporation. The crude product was then
rinsed and filtered with cold ethyl acetate, the solvent removed by
rotary-evaporation and the process repeated in cold methanol to remove
all solid impurities. The product was then purified by column
chromatography and fluorous chromatography (Fluorous Technologies).
Spectral data: 1H NMR (CDCl3) δ: 3.98 (t, 2H), 3.74 (t,
2H), 3.67 (t, 2H), 3.62 (m, 6H), 3.55 (t, 2H), 3.0 (b, 1H); 13C NMR
(CDCl3) δ: 120-105, 72.65, 72.44, 70.86, 70.64, 70.43, 68.35
(t, J=100 Hz), 61.73. Electrospray mass spectrometry in positive mode
showed a single peak at 583.0 m/z corresponding to
[n-C8F17CH2-OEG6+H+]+.

Synthesis of n-C8F17CH2-OEG6

[0060] Hexaethylene glycol (Sigma-Aldrich, MW: 282.33) was dried over
molecular sieves and 1 equivalent was added to a dried round bottom flask
with stir bar. 2.5 equivalents of CF3(CF2)7CH2OH
(Sigma-Aldrich, MW: 450.1) and 1.5 equivalents of
1,1'-azodicarbonyldipiperidine (ADDP) (TCI, MW: 252.31) were also added
to the flask. These were dissolved in anhydrous benzene (so that
concentrations were around 0.1 M). The reaction mixture was then stirred
at room temperature. Then 1.6 equivalents of tributylphosphine
(Sigma-Aldrich, MW: 202.32) was dissolved in benzene (about half the
volume that was added to the round bottom flask) in an addition funnel.
This solution was then slowly added to the reaction mixture over 3-4
hours, and the reaction allowed to run overnight. The precipitant was
filtered off and solvent removed by rotary-evaporation. The crude product
was then rinsed and filtered with cold ethyl acetate, the solvent removed
by rotary-evaporation and the process repeated in cold methanol to remove
all solid impurities. The product was then further purified by column
chromatography and fluorous chromatography. Spectral data: 1H NMR
(CDCl3) δ: 4.02 (t, 2H), 3.74 (t, 2H), 3.68 (t, 2H), 3.61 (m,
18H), 3.56 (t, 2H), 3.0 (b, 1H); 13C NMR (CDCl3) δ:
120-105, 72.65, 72.37, 70.76, 70.72, 70.67-70.59, 70.37, 68.33 (t, J=100
Hz), 61.73. Electrospray mass spectrometry in positive mode showed a
single peak at 715.0 m/z corresponding to [(1)+H+]+.

[0062] RfNTA is synthesized by attaching a nitrilotriacetate (NTA) head
group to n-C8F17CH2-OEG6 (Compound 1, See Scheme 1).
n-C8F17CH2-OEG6 was synthesized as described herein
using Mitsunobu conditions. 10 equivalents of
1,8-Diazabicyclo[5.4.0]undec-7-ene (Acros, MW: 152.24) and 1 equivalent
of Nα,Nα-Bis(carboxymethyl)-L-lysine trifluoroacetate salt
(Compound 3) (Sigma-Aldrich, MW:376.3) was suspended in dry methylene
chloride. Then Compound 2 (MW: 808.5) was dissolved in methylene chloride
and added to the reaction mixture and the reaction was stirred overnight
at room temperature under dry nitrogen. The solvent was then removed and
the remaining solid washed several times with EA. The solid was then
dissolved in a minimal amount of methanol (Fisher) and sodium
isopropylphenoxide (prepared by mixing sodium methoxide (Acros) with a
slight excess of isopropylphenol (Sigma-Aldrich)) was added. The mixture
was stirred for 15 minutes and any solid filtered off. The liquid was
then evaporated, the solid dissolved in a minimal amount of methanol and
the product was precipitated using ether (Fisher). The product Compound 4
(MW=1068.6) was then further purified using fluorous chromatography.
Spectral data: 1H NMR (CD3OD) δ: 4.1 (m, 4H), 3.7 (m,
5H), 3.61 (m, 22H), 3.10 (m, 2H), 2.0-1.5 (m, 6H); 13C NMR
(CDCl3) δ: 158.94, 129.93, 73.165, 71.49-71.28, 70.85, 69.31,
68.97 (t, J:=100 Hz), 64.85, 56.86, 41.28, 30.52, 28.43, 25, 48.
Electrospray mass spectrometry in negative mode showed a predominant peak
at 1000.8 m/z, and an additional peak at 1023.8 m/z consistent with
[RfNTA-3Na++2H+].sup.- salt and [RfNTA-2Na+H+]respectively. It will be apparent to one skilled in the art that RfNTA
can be converted to an acid, or can be isolated as a partially or fully
deprotonated compound together with singly or multiply charged cations.

[0064] PDMS was used to fabricate all microfluidic devices. Microchannels
with rectangular cross-sections were fabricated with rapid prototyping as
described elsewhere herein. The hybrid microfluidic devices consisted of
PDMS microchannels (cross-section: 250 μm by 250 μm) with three or
four tapered inlets (100 μm by 250 μm) for aqueous phases and one
orthogonal inlet for carrier fluid to form nanoliter plugs. The channel
walls were functionalized with
(tridecafluoro1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (United Chemical
Technologies) to render them hydrophobic and fluorophilic. Teflon tubing
was inserted into the outlet up to the plug forming junction. The tubing
was sealed to the device with either wax or PDMS glue.

Interfacial Adsorption Experiments Using hGFP.

[0065] Three-aqueous-inlet devices were used to perform interfacial
adsorption experiments. In these experiments the left aqueous stream
contained the additive solution, which was either (1) 100 mM Tris, pH
7.0, (2) 115 μM NiSO4 in 100 mM Tris, pH 7.0, (3) 125 μM RfNTA
in 100 mM Tris, pH 7.0, (4) 125 μM Compound 3 and 115 μM NiSO4
in 100 mM Tris, pH 7.0, or (5) 62.5 μM RfNTA and 57.5 μM NiSO4
in 100 mM Tris, pH 7.0. The stock buffer solution had been pretreated
with Chelex to remove any divalent cations and all other solutions were
made from this. When mixed with RfNTA the concentration of Ni2+ was
always between 90-95% of the RfNTA concentration to ensure no free
Ni2+ was left in solution. Higher concentrations of RfNTA:Ni were
also tested and performed similarly. The center stream always contained
100 mM Tris, pH 7.0 except for an experiment with 20 mM EDTA (Fisher) in
100 mM Tris, pH 7.0. The right stream contained 2 μM hGFP (Upstate,
now part of Millipore) and 0.25% lauryldimethylamine N-oxide (LDAO)
(Anatrace) (w/v) in 100 mM Tris, pH 7.0. Plugs were formed into Teflon
tubing (OD 150 μm, ID 50 μm) using FC-40 that had been
pre-equilibrated with 20 mM EDTA. The carrier fluid flowed at a rate of
8.3-16.67 nL/sec, using PHD2000 syringe pumps (Harvard Apparatus). No
additional surfactant was used in the FC-40 except when 0.25 mg/mL
n-C8F17CH2-OEG3 (MW 582.25) was used. The hGFP always
flowed at 3.33 nL/sec so the final concentration was 400 nM hGFP and
0.05% LDAO. The other two flow rates were adjusted so the total aqueous
flow rate was always 16.67 nL/sec. The additive and buffer flow rates
were both 6.67 nL/sec for additive conditions 1-4, so the final
concentrations were 46 μM Ni2+, 50 μM RfNTA, and 50 μM
Compound 3 and 46 μM Ni2+, for conditions 2-4 respectively. For
condition 5 the flow rates were 3.33 and 10 nL/sec for the additive and
buffer stream leading to final concentrations of 12.5 μM RfNTA and
11.5 μM Ni2+. When EDTA was added to the buffer stream it gave a
final concentration of 12 mM EDTA. Plugs were formed for at least 2
minutes, then flow was stopped, the tubing cut and ends sealed onto a
glass slide with wax. For imaging the tubing was submerged in
milliQ-H2O. Bright field and fluorescence images were obtained using
confocal microscopy on a Leica DMI6000 microscope (Leica Microsystems)
using a VT-Infinity 3 confocal scanning head (VisiTech international).
They were analyzed using SimplePCI software (Hammamatsu Corp.), and
MetaMorph 6.3 (Molecular Devices).

FRAP Experiments.

[0066] Plugs were formed as described elsewhere herein. The final
concentration of His 10 was 3.7 μM in 20 mM Tris pH 8.0. The final
RfNTA:Ni concentration was 30 μM RfNTA and 28.5 μM Ni. Plugs were
formed in three different fluorocarbons: FC-70 with a viscosity of 12
cSt, FC-40 with a viscosity of 1.8 cSt, and FC-84 with a viscosity of
0.53 cSt at room temperature. All fluorocarbons contained 170 μM
n-C8F17CH2-OEG3 to prevent nonspecific adsorption.

[0067] During the FRAP experiments 10 images of the edges of a plug were
obtained before bleaching. Next, spots were bleached on the edges of the
plugs using high laser power for 2 seconds, and the regions were then
monitored for an additional 40 seconds. Linescans spanning the edges of
the plugs were used to monitor fluorescence recovery at the plug
interface over time. The shape of the bleach spot was fit using Origin 8
(OriginLab) to the Gaussian curve described by Equation 1. A generalized
solution to Fick's second law of diffusion is given in Equation 2 and
draws obvious parallels to Equation 1 (See Seiffert, S, and Oppermann,
W., J. Microsc.--Oxf. 2005, 220, 20-30).

[0068] I is intensity, I0 is the initial intensity, A(t) is the
maximum amplitude at time t, x is the position, and x0 is the
offset, and w determines the distribution width of the curve. C is the
concentration, C0 is the initial concentration. M is the total
amount of the diffusing species, D is the diffusion coefficient, r is the
position, and t is time.

[0069] Monitoring the change in the shape of the Gaussian over the time
course of recovery allows for extraction of a diffusion coefficient and
requires no predetermined knowledge of the geometry of the bleach spot or
of the diffusion dimension (See Seiffert, S, and Oppermann, W., J.
Microsc.--Oxf. 2005, 220, 20-30). From Equations 1 and 2 it can be seen
that w2=2*D*t, so plotting w2 vs. t should give a straight line
with slope 2*D. The diffusion dimension can be determined using log
A(t)=-d/2*log(t)+K.

[0070] This analysis gave diffusion coefficients of
7.3±2.6×10-12 m2/sec, 15.2±2.4×10-12
m2/sec, 39.2±11.5×10-12 m2/sec for FC-70, FC-40
and FC-84 respectively. In all cases there was substantially complete
recovery of fluorescence indicating that no immobile phase existed. The
measured diffusion dimension was 1.1±0.3, 1.1±0.2, 2.0±0.4 for
FC-70, FC-40 and FC-84 respectively. However, due to the log-log plot,
the accuracy of d is greatly affected by the time range in which reliable
data can be obtained. Divalent metals are capable of quenching
fluorescein, and significant quenching was observed when using His10.
This led to fairly noisy data, which limited the timescale over which
accurate measurements could be obtained. Analysis that included later
time points indicated that d for FC-70 was likely accurate; whereas d for
FC-40 fell between 1 and 2, and d for FC-84 remains near 2. This general
trend of increasing diffusion dimension with faster diffusion can be
explained if the geometry of the bleaching zone is considered. The
bleaching zone is not a point; rather, there is a cone above and below
the focal plane. If the bleaching zone of the laser above and below the
focal plane is large compared to the length scale of diffusion, then the
predicted diffusion dimension would be 1, as recovery can only come from
fluorophore in the focal plane. On the other hand, for faster diffusion,
the diffusion dimension would approach 2 as fluorophores above and below
the plane can contribute.

[0071] Similar experiments were performed with hGFP at 1 μM, and
fluorescein-labeled hSA at 250 nM. The hGFP could not be analyzed in the
same fashion due to a quenching effect that resulted in a temporary
enhancement of fluorescence around the bleach spot during recovery. Rough
estimates of Do, obtained by fitting fluorescence recovery of the
area surrounding the bleach spot with a single exponential curve, were
approximately 3×10-12 m2/sec for FC-40 and 30 μM
RfNTA:Ni. For the labeled hSA interfacial binding was weak, possibly due
to the close proximity of the His-tag to structural components of the
protein. However, initial experiments resulted in D0 using the
Gaussian fitting model of 4.5±0.6 and 9.2±2.4×10-12
m2/sec for FC-70 and FC-40, respectively. Without wishing to be
bound by theory, the hGFP and hSA are thought to give slower diffusion
than His10 because a protein would create more drag than a small peptide.
However the D0 for a protein in solution is around
1×10-10 m2/sec, so events at the interface still dominate
the diffusion rather than the attached protein or peptide.

[0072] FRAP was measured using a Leica tandem scanner SP5 spectral
confocal on a DMI6000 microscope with a 40×NA1.40 oil objective.
Images were analyzed by using ImageJ software and the plug-in loci
bio-formats and by using Mathematica.

Tensiometry.

[0073] Droplets of fluorous surfactant solution were formed at the end of
disposable droplet extrusion tips. The tips were assembled by using
quick-set epoxy to glue polyimide-coated glass tubing into 1-10 μL
disposable pipette tips that were oxidized in a Plasma Prep II plasma
cleaner (SPI Supplies) for 3 min to render them hydrophilic. The end of
the capillary was positioned just inside the end of the pipette tip. The
polyimide tubing was connected to a 50 μL Hamilton Gastight syringe
using 30-gauge Teflon tubing. The syringe was filled with the fluorous
solution. The pipette tip was positioned so that it sat in the aqueous
solution within a 1 mL polystyrene cuvette and held in place by a clamp.
The formed droplets were imaged using Model 250 Standard Digital
Goniometer & DROPimage Advanced software (Rame-Hart Instrument Co).

Crystallization of Reaction Center (RC).

[0074] Four-aqueous-inlet devices were used to perform crystallization
experiments. In sequence, the precipitant stream was 50% (w/v) PEG 4000,
1.1 M NaCl, 9% (w/v) 1,2,3-heptanetriol (HPT) in 50 mM Tris pH 7.8. The
buffer stream was 9% HPT in 50 mM Tris pH 7.8. The additive stream used
six different solutions in six different sets of experiments, and they
were (1) 10 mM Tris pH 7.8, which was defined as the standard condition,
(2) 45 μM NiSO4 in 10 mM Tris pH 7.8, (3) 50 μM RfNTA in 10 mM
Tris pH 7.8, (4) 50 μM RfNTA and 45 μM NiSO4 in 10 mM Tris pH
7.8, (5) 200 μM RfNTA and 180 μM NiSO4 in 10 mM Tris pH 7.8,
and (6) 200 μM RfNTA and 180 μM NiSO4 and 10 mM imidazole in
10 mM Tris pH 7.8. The protein stream was 6 mg/mL hRC from Rhodobacter
sphaeroides in 0.05% (w/v) LDAO and 10 mM Tris pH 7.8. The fluorinated
carrier fluid was FC-40. The carrier fluid, protein, and additive streams
were maintained at constant flow rates of 41.7 nL/sec, 13.3 nL/sec and
3.3 nL/sec, respectively. The flow rate of the precipitant stream was
first increased from 8.3 nL/sec to 15 nL/sec, and then decreased from 15
nL/sec to 8.3 nL/sec, with a step size of 1.7 nL/sec. Correspondingly,
the buffer stream was first decreased from 8.3 nL/sec to 1.7 nL/sec, and
then increased from 1.7 nL/sec to 8.3 nL/sec, with a step size of 1.7
nL/sec. Each flow rate step lasted for 14 s. After one step was finished,
the subroutine was stopped and, ˜5 s later, it was restarted with
the setup of the next step. In this experiment, two sets of plugs
generated from identical flow rates were counted as duplicates. The
experiments were performed six times with six different additive
solutions. The trials, in the form of plugs, were transported and stored
in Teflon tubing (O.D.: 250 μm and I.D.: 200 μm) which was sealed
inside glass tubing (O.D.: 3 mm and I.D.: 1.8 mm) prefilled with FC-70.
The experiment was performed under dim light, and the trials were kept in
the dark at 23° C.

[0076] Plugs were counted under dim light at day 1, day 2, day 5 and day
12 after setting up the experiments. In all experiments for each flow
rate step, 40 plugs were counted starting from the tenth plug of each
set. Plugs with crystals were counted as one hit. To make sure one did
not start from different plugs at different days, a picture of the first
counted plug in each set of plugs was taken.

Determining the Nucleation Rate.

[0077] The nucleation rate was determined for different additive
conditions at two precipitant concentrations: (1) Concentration B, where
plugs of crystallization trials were formed at 13.3 nL/sec and (2)
Concentration A, where plugs of crystallization trials were formed at 15
nL/sec. In (1) final concentrations of precipitant were 20% (w/v) PEG
4000, 0.44 M NaCl and 30 mM Tris pH 7.8, whereas in (2) final
concentrations of precipitant were 22.5% (w/v) PEG 4000, 0.5 M NaCl and
30 mM Tris pH 7.8. To determine the nucleation rate for each
concentration of precipitant, the percentage of the number of plugs with
crystals out of the total number of counted plugs (40 in all cases) was
plotted against the recording time (days). The nucleation rate was then
extracted from the slope of the plots, determined by dividing the change
in the number of plugs with crystals by the change in time. The
nucleation rate was equal to the largest slope for each curve. If no
crystals were obtained over time, the nucleation rate was zero. All the
nucleation rates were extracted from day 0 to day 1 except the one for
the standard condition at Concentration A, which was extracted from day 0
to day 5.

[0078] In these experiments, the number of plugs were counted instead of
the number of crystals. For standard conditions, there was always only
one crystal per plug, whereas many of the other conditions often had
multiple crystals per plug. Because the number of individual nucleation
events was not counted, the calculated nucleation rates represent the
lower limit of the actual nucleation rate.

Crystal Preparation and X-Ray Data Collection.

[0079] Cryo-protectant for freezing hRC crystals was 25% (v/v) PEG 400,
20% (w/v) PEG 4000, 0.44 M NaCl in 0.08% (w/v) LDAO and 20 mM Tris pH
7.8. Crystals of hRC from R. sphaeroides grown in the plugs were
extracted by attaching a syringe to one end of the Teflon tubing and
flowing the crystals slowly into a drop of cryo-protectant by using the
manual syringe driver. Once crystals were flowed into a 2 μL
cryo-protectant drop, the crystals were picked up with a CryoLoop
(Hampton Research) and flash frozen in liquid nitrogen. The X-ray
diffraction experiments were performed at GM/CA Cat station 23 ID-B of
the Advanced Photon Source (Argonne National Laboratory). In all
experiments, the wavelength was kept at 1.03 Å. A 10 μm minibeam
was used with the attenuation at five. The exposure time was kept at 10
seconds. The data were processed in HKL2000 to determine the space group
and diffraction limit with S/N over 2.0. Crystals from 200 μm RfNTA:Ni
at Concentration B diffracted best to 3.1 Å; b: Crystals from the
standard condition at Concentration A diffracted best to 4 Å and c:
Crystals from 50 μM RfNTA at Concentration A diffracted best to 4
Å.

[0080] From the foregoing, it will be observed that numerous variations
and modifications may be effected without departing from the spirit and
scope of the invention. It is to be understood that no limitation with
respect to the specific embodiment illustrated herein is intended or
should be inferred. It is, of course, intended to cover by the appended
claims all such modifications as fall within the scope of the claims.